Plasma membrane phospholipids form stoichiometric complexes with cholesterol. Excess cholesterol beyond the binding capacity of the phospholipids creates a novel pool of cholesterol with a high chemical activity. This high-activity cholesterol then serves as a homeostatic signal; for example, by regulating endoplasmic reticulum effectors of cell cholesterol balance and mitochondrial oxysterol biosynthesis. The present proposal will consider two additional and unexplored regulatory roles for active plasma membrane cholesterol; namely, in mitochondrial steroidogenesis and in the transfer of plasma membrane cholesterol to circulating high density lipoproteins by the scavenger receptor SR-BI and the ABCA1 transporter. A novel hypothesis for the mysterious molecular mechanism of general anesthesia is also proposed. We suggest that a wide range of anesthetics act by intercalating into the plasma membrane bilayer of neurons, displacing and thereby activating a small fraction of their cholesterol. This pool of active cholesterol would then perturb neuronal ion channels, leading to anesthesia. This hypothesis will be tested by examining the influence on ion channel activity of a library of amphipaths that activate cholesterol. The effects of cholesterol itself and of cholesterol-complexing phospholipids on ion channel activity will also be tested. We note that the function of numerous plasma membrane proteins is dependent on or otherwise influenced by cholesterol. The premise that these phenomena are related to cholesterol-driven lateral phase separation (i.e., raft formation) is powerful but controversial. We shall therefore characterize diverse water-soluble intercalating amphipaths as probes that can increase, decrease or otherwise perturb the formation of putative plasma membrane rafts, allowing for both their analysis and manipulation. These probes act by complexing with bilayer phospholipids, displacing and substituting for their cholesterol so as to alter the driving force for lateral phase partition. Intercalated amphipaths that modulate raft/microdomain formation and behavior could have basic scientific and perhaps therapeutic utility. These studies will test new hypotheses about the physiology of active cholesterol. They will greatly increase our fundamental understanding of this essential membrane component, bearing on varied and important medical issues, such as cardiovascular disease, (patho)physiological processes mediated by raft-embedded proteins and clinical anesthesia. PUBLIC HEALTH RELEVANCE: Cell cholesterol is of universal interest and concern because of the enormous burden on health imposed by atherosclerotic cardiovascular disease. It is widely appreciated that atherogenesis is to be understood as normal physiological processes gone awry and that insights are needed into the basic sterol management systems inherent in eukaryotic cells. Elucidating the physiology and practical implications of membrane cholesterol complexes and cholesterol activity will hopefully play a role in the development of therapeutic and preventive approaches to cholesterol-related diseases. .